Psychological stress is associated with increased risk of several health conditions including cardiovascular disease, autoimmune disorders, infectious disease, and mental illnesses (B. S. McEwen, Stress, adaptation, and disease. Allostasis and allostatic load, Ann. N. Y. Acad. Sci. 840 (1998) 33-44). The link between psychological stress and physical ailments can be observed in the biological responses associated with stress, namely the production of cortisol, a major glucocorticoid in humans.
Cortisol is synthesized and secreted by the zona fasciculata and the zona reticularis of the adrenal cortex for the purpose of facilitating the human body to adapt to changing environmental conditions (H. Dobson, and R. F. Smith, “What is stress, and how does it affect reproduction?,” Animal reproduction science, vol. 60, pp. 743-752, 2000). In its free form, cortisol plays an important role in the regulation of, for example, blood pressure, glucose levels, carbohydrate metabolism, and homeostasis of the cardiovascular, immune, renal, and endocrine systems (E. R. De Kloet, M. Joëls, and F. Holsboer, “Stress and the brain: from adaptation to disease,” Nature Reviews Neuroscience, vol. 6, pp. 463-475, 2005.; R. Gatti, G. Antonelli, M. Prearo, P. Spinella, E. Cappellin, and F. Elio, “Cortisol assays and diagnostic laboratory procedures in human biological fluids,” Clinical biochemistry, vol. 42, pp. 1205-1217, 2009; R. Fraser, M. C. Ingram, N. H. Anderson, C. Morrison, E. Davies, J. M. C. Connell, Cortisol Effects on Body Mass, Blood Pressure, and Cholesterol in the General Population, Hypertension, 33, 1364-1368, 1999; D. S. Charney, Psychobiological Mechanism of Resilience and Vulnerability: Implications for Successful Adaptation to Extreme Stress, Am. J. Psychiatry, 161, pp. 195-216, 2004).
Any abnormality in cortisol levels inhibits inflammation, depresses the immune system, and increases fatty and amino acid levels in blood. Persistent increment of cortisol may affect brain functions leading to aging of the brain. A decrease in cortisol levels contributes to the development of Addison's disease having symptoms such as weight loss, fatigue, and darkening of skin folds and scars, while elevated cortisol levels can lead to Cushing's disease with the symptoms of obesity, fatigue, and bone fragility (O. M. Edwards, J. M. Galley, R. J. Courtenay-Evans, J. Hunter, and A. D. Tait, “Changes in cortisol metabolism following rifampicin therapy,” The Lancet, vol. 304, pp. 549-551, 1974; B. S. McEwen, “Cortisol, Cushing's syndrome, and a shrinking brain—new evidence for reversibility,” The Journal of Clinical Endocrinology & Metabolism, vol. 87, pp. 1947-1948, 2002). Cortisol levels can increase by ten-fold following surgery or other major trauma, as the steroid acts to prevent vascular collapse, reduce inflammation, and suppress immune response. Chronically elevated cortisol is associated with the neuroendocrine causal pathway linking environmental or psychological distress to poor health outcomes (M. van Eck, H. Berkhof, N. Nicolson, J. Sulon, The effects of perceived stress, traits, mood states, and stressful daily events on salivary cortisol, Psychosom. Med., 58, pp. 447-58). Hence, cortisol is considered a major stress hormone and is, therefore, an effective biomarker for stress.
Existing cortisol detection techniques are generally limited to the conventional laboratory-based techniques such as chromatography (B. J. Klopfenstein, J. Q. Purnell, D. D. Brandon, L. M. Isabelle, and A. E. DeBarber, “Determination of cortisol production rates with contemporary liquid chromatography—mass spectrometry to measure cortisol-d3 dilution after infusion of deuterated tracer,” Clinical Biochemistry, 44, pp. 430-434, 2011; L. Q. Chen, X. J. Kang, J. Sun, J. J. Deng, Z. Z. Gu, and Z. H. Lu, “Application of nanofiber—packed SPE for determination of salivary—free cortisol using fluorescence precolumn derivatization and HPLC detection,” Journal of separation science, 33, pp. 2369-2375, 2010; W. Gao, Q. Xie, J. Jin, T. Qiao, H. Wang, L. Chen, H. Deng, and Z. Lu, “HPLC-FLU detection of cortisol distribution in human hair,” Clinical biochemistry, vol. 43, pp. 677-682, May 2010), radioimmunoassay (MA) (D. Appel, R. D. Schmid, C. A. Dragan, M. Bureik, and V. B. Urlacher, “A fluorimetric assay for cortisol,” Analytical and bioanalytical chemistry, 383, pp. 182-186, 2005), electrochemiluminescence immunoassay (ECLIA) (H. Shi, X. Xu, Y. Ding, S. Liu, L. Li, and W. Kang, “Determination of cortisol in human blood sera by a new Ag (III) complex-luminol chemiluminescent system,” Analytical biochemistry, 387, pp. 178-183, 2009), enzyme-linked immunosorbent assay (ELISA) (J. G. Lewis, and P. A. Elder, “An enzyme-linked immunosorbent assay (ELISA) for plasma cortisol,” Journal of steroid biochemistry, 22, pp. 673-676, 1985; L. Manenschijn, J. W. Koper, S. W. J. Lamberts, and E. F. C. van Rossum, “Evaluation of a method to measure long term cortisol levels,” Steroids, 76, pp. 1032-1036, 2011; M. Shimada, K. Takahashi, T. Ohkawa, M. Segawa, and M. Higurashi, “Determination of salivary cortisol by ELISA and its application to the assessment of the circadian rhythm in children,” Hormone Research in Paediatrics, 44, pp. 213-217, 1995), surface plasmon resonance (SPR) (J. S. Mitchell, T. E. Lowe, and J. R. Ingram, “Rapid ultrasensitive measurement of salivary cortisol using nano-linker chemistry coupled with surface plasmon resonance detection,” Analyst, 134, pp. 380-386, 2008; D. R. Shankaran, K. V. Gobi, and N. Miura, “Recent advancements in surface plasmon resonance immunosensors for detection of small molecules of biomedical, food and environmental interest,” Sensors and Actuators B: Chemical, 121, pp. 158-177, 2007), quartz crystal microbalance (QCM) (M. Z. Atashbar, B. Bejcek, A. Vijh, and S. Singamaneni, “QCM biosensor with ultra thin polymer film,” Sensors and Actuators B: Chemical, 107, pp. 945-951, 2005), and piezoelectric immunosensor (B. S. Attili, and A. A. Suleiman, “A piezoelectric immunosensor for the detection of cortisol,” Polymer—Plastics Technology and Engineering, 28, pp. 2149-2159, 1995). These laboratory techniques are not only expensive, laborious, and time-consuming, but also often require complex systems, assay formation complexity, large sample volume, and time-consuming incubation and separation procedures, which altogether limit them being adapted in point-of-care (POC) applications. Although several electrochemical immunosensing platforms have been developed recently for POC cortisol detection, they often require complex synthesis and fabrication steps involving the immobilizing matrix of high surface functionality, high biomolecule loading, and small resistance to electron transport (A. Kaushik, A. Vasudev, S. K. Arya, S. K. Pasha, and S. Bhansali, “Recent advances in cortisol sensing technologies for point-of-care application,” Biosensors and Bioelectronics, 53, pp. 499-512, 2014; P. K. Vabbina, A. Kaushik, N. Pokhrel, S. Bhansali, and N. Pala, “Electrochemical cortisol immunosensors based on sonochemically synthesized zinc oxide 1D nanorods and 2D nanoflakes,” Biosensors and Bioelectronics, 63, pp. 124-130, 2015; P. K. Vabbina, A. Kaushik, K. Tracy, S. Bhansali, and N. Pala, “Zinc oxide nanostructures for electrochemical cortisol biosensing,” Proc. SPIE, 9107, pp. 91070U-91070U, 2014).
Additionally, a major limitation of currently available cortisol immunoassay kits and immunosensors is their cross-reactivity and interference with other cortisol structural analogs such as, for example, progesterone and prednisolone (S. Tunn, G. Pappert, P. Willnow, M. Krieg, Multicentre evaluation of an enzyme-immunoassay for cortisol determination., Clin. Chem. Clin. Biochem., 28, pp. 929-35, 1990; I. A. Ionita, D. M. Fast, F. Akhlaghi, Development of a sensitive and selective method for the quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma., J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 877, pp. 765-72, 2009).